Same analytical method for both (bio)assay and zone isolation to identify/quantify bioactive compounds by quantitative nuclear magnetic resonance spectroscopy

Differing sensitivity is the main obstacle for a direct combination of HPTLC with NMR spectroscopy. A sufficient amount of the isolated compound zone must be provided by HPTLC for subsequent offline NMR detection (HPTLC//NMR). To fill the gap, a straightforward procedure was developed using the same analytical HPTLC system for both bioprofiling and isolation of bioactive zones from multicomponent mixtures. The HPTLC-effect-directed analysis (EDA) revealed several bioactive compounds in five botanical extracts, i.e. Salvia officinalis, Thymus vulgaris and Origanum vulgare, all Lamiaceae, and peels of red and green apples (Jonagored and Granny Smith, respectively), both Rosaceae. A tricky case study was designed to show how to deal with potentially coeluting bioactive structural isomers, e.g., ursolic (UA), oleanolic (OA) and betulinic acids (all C₃₀H₄₈O₃), which are most difficult to identify and assign. A multipotent bioactive HPTLC zone showed the same hR_F value and mass signal in HPTLCHRMS, though containing the coeluting structural isomers UA and OA. After zone isolation from the HPTLC plate, first the ¹H NMR spectrum allowed to distinguish distinct allylic H-18 protons, i.e. 2.20 ppm for UA and 2.85 ppm for OA, and at the same time, to quantify the two isomers by using the PUlse Length-based CONcentration methodology (HPTLC//¹H qNMR-PULCON). In case of a partial overlap of the diagnostic signal with that of the matrix, results were corroborated with those obtained by using the ¹H deconvoluted or 2D ¹H-¹³C Heteronuclear Single Quantum Coherence spectra. The comparison of the quantitative results showed a good correlation (R² = 0.9718) between the two orthogonal methods HPTLC-Vis and HPTLC//¹H qNMR-PULCON. A sufficient zone isolation from the HPTLC plate (mean isolation rate of 82%) for both UA and OA (0.27 - 4.67 mM) was achieved for HPTLC//qNMR, comparing the isolated bioactive compound zone with the respective zone in the botanical extract via HPTLC-Vis densitometry. The HPTLC-EDA-Vis//¹H qNMR-PULCON procedure for bioprofiling and quantification/identification/confirmation of bioactive compounds in botanical extracts is considered as straightforward, eco-friendly (only 16 mL solvent required), simple (NMR calibration used over weeks) and reliable new alternative to the status quo of bioactivity-guided fractionation.

Keywords: Biochemical or biological assay; HPTLC PULCON; Hydroxy pentacyclic triterpenoic acid isomers; Quantitative NMR (qNMR); Sage, thyme, origano and apple.